JP2004500815A - Methods for obtaining proteins with improved functional properties - Google Patents

Abstract

An improved cellulase comprising an amino acid sequence which has been modified from a precursor amino acid sequence by the substitution or deletion of an amino acid residue which differs from a corresponding amino acid residue in a less stable but homologous cellulase, wherein said improved cellulase has improved thermostability compared to a cellulase corresponding to the precursor amino acid sequence.

Description

【００３６】
ｐＢＬａｐｒプラスミドを、Ｂａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓのα−アミラーゼに関する遺伝子を含むように構築した。図７で説明されているように、ｐＢＬａｐｒは、ｐＢＲ３２２からのアンピシリン耐性遺伝子およびｐＣ１９４からのクロラムフェニコール耐性遺伝子、ａｐｒＥプロモーターおよびＢａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓのα−アミラーゼをコードする遺伝子（「ＢＬ　ＡＡ」）を含む、６．１ｋｂのプラスミドである。ａｐｒＥプロモーターは、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓのアルカリプロテアーゼのプロモーターおよびシグナル配列をコードする６６０ｂｐのＨｉｎｄＩＩＩ−ＰｓｔＩフラグメントで構成された。Ｐｓｔｌ部位を除去し、ＳｆｉＩ部位をａｐｒＥ／ＢＬ　ＡＡ結合部の近くに付加した。ＢＬ　ＡＡ遺伝子は、上述した１７２０ｂｐのＰｓｔＩ−ＳｓｔＩフラグメントに含まれる。ここで説明される実験において、ｐＢＬａｐｒは、成熟アミラーゼに関するコード配列の開始部位の５’末端に隣接するＳｆｉＩ部位で構成されている。特に、ｐＢＬａｐｒ構造の５’末端は、ｐＢＬａｐｒからＭ１３ＢＭ２０（ベーリンガーマンハイム社）へ、ＥｃｏＲＩ−ＳｓｔＩＩフラグメントをサブクローニングし、以下に示す変異オリゴヌクレオチドに関するコード鎖の鋳型を得た。
【００３７】
【化２】

【００３８】
このプライマーをＳｆｉＩ部位（下線で示す）に導入した。それにより、この特定の制限部位が存在することによってスクリーニングされ得る正しい形態にした。ＥｃｏＲＩ−ＳｓｔＨフラグメントをｐＢＬａｐｒベクターへサブクローニングにより戻すことにより、ＳｆｉＩ部位を有するプラスミドのバージョン（ｖｅｒｓｉｏｎ）を得た。
【００３９】
プラスミドｐＨＰ１３（Ｈａｉｍａ ｅｔ ａｌ．， Ｍｏｌ． Ｇｅｎ． Ｇｅｎｅｔ．， ＶＯｌ． ２０９， ｐｐ．３３５−３４２ （１９８７））（図６）を、制限酵素ＥｃｏＲＩおよびＨｉｎｄＩＩＩで消化し、得られたベクターを、ポリアクリルアミドゲル上で精製し、溶離した。プラスミドｐＥｈａｐｒを、ＨｉｎｄＩＩＩおよびＡｓｐ７１８で消化し、別のインキュベーションで、Ａｓｐ７１８およびＥｃｏＲＩで消化し、ゲルで精製した。二つのバンド、すなわちＨｉｎｄＩＩＩ−Ａｓｐ７１８（１２０３ｂｐ）およびＡｓｐ７１８−ＥｃｏＲＩ（１２５３ｂｐ）をゲルで精製し、ゲルから溶離し、３ウェイライゲーション（３−ｗａｙ ｌｉｇａｔｉｏｎ）によってベクターに結合し、プラスミドｐＨＰ．ＢＬを得た。当該プラスミドをα−アミラーゼの発現に用いた（図８）。
【００４０】
実施例２
アスパラギン１８８に関する置換を含むα−アミラーゼをコードするプラスミドの構築
天然に存在する各アミノ酸によるＡｓｎ１８８（「Ｎ１８８」）の置換をコードする変異プライマー群を合成し、そして図１に示す（配列番号４〜２２）。これらの変異をコードするα−アミラーゼ遺伝子変異体を、図９に要約される方法によって、図２に示すＰＣＲプライマー（配列番号２３〜３２）を用いてＰＣＲで製造した。
【００４１】
段階（１）：変異プライマーを、ＰＣＲプライマーのＰＣＲ　Ａ＋およびＰＣＲ　Ｂ−のためのテンプレートとして用い、伸長した（６１ｂｐ）二重鎖ＤＮＡを得た。それぞれ、１８８位に異なるアミノ酸の置換を含み、Ｎ１８８Ｍ以外は全て、異なる制限部位を含んだ。はじめに、ＰＣＲプライマーを、３５℃で５分間アニールし、続いてＴａｑポリメラーゼにより７５℃で１分間ＤＮＡ伸長した。次に二重鎖ＤＮＡを９５℃で１分間解離し、続いてアニーリング段階および伸長段階を行った。解離、アニーリングおよび伸長のサイクルを、総計３０サイクル続けた。
【００４２】
段階（２）：１８８位の上流および下流のＤＮＡを、それぞれ別のＰＣＲ反応で合成した。テンプレートとしてはｐＢＬａｐｒを用い、ＰＣＲプライマーとしては、上流のＤＮＡに関してはＬＡＡｆｓ５（配列番号２７）およびＰＣＲ　Ａ−（配列番号２４）を用い、下流のＤＮＡに関してはＰＣＲ　Ｂ＋（配列番号２５）およびＰＣＲ　Ｃｌａ−ＳａｌＩ（配列番号２８）を用いた。ＤＮＡを９５℃で１分間解離し、４５℃で３分間アニールし、６８℃で３分間伸長させた。上流の部分は２９０ｂｐであり、下流は４９８ｂｐである。この方法を、ｐｆｕポリメラーゼを用いて１８サイクル繰り返した。同じＰＣＲ方法を、段階（３）および（４）で用いた。
【００４３】
段階（３）：段階（２）で説明したＤＮＡの上流部分を、段階（１）で説明した二重鎖の変異プライマーに付加した。プライマーＬＡＡｆｓ５（配列番号２７）およびＰＣＲ　Ｂ−（配列番号２６）を用いた。３つのテンプレート間で２４ｂｐのオーバーラップが生じるようにプライマーを設計し、ここに２ピースのＤＮＡを付着させることができる。
【００４４】
段階（４）：段階（２）で示されたＤＮＡの下流部分と、段階（３）の生産物とを結合して、目的の生産物を得た。２つのＰＣＲ産物間にある２４ｂｐのオーバーラップにより、結合が可能である。用いられたプライマーは、ＬＡＡｆｓ５（配列番号２７）およびＰＣＲ　ＣｌａＩ−ＳａｌＩ（配列番号２８）であった。
【００４５】
段階（５）：特異的な制限部位、すなわちＡｓｐ７１８およびＢｓｓＨＩＩは、それぞれ１８８部位の上流および下流に存在する。目的のＰＣＲ産物は、Ａｓｐ７１８およびＢｓｓＨＩＩで分解され、３３３ｂｐのフラグメントをポリアクリルアミドゲル電気泳動によって分離し、ｐＨＰ．ＢＬベクターにサブクローニングし、ｐＨＰ．Ｎ１８８Ｘを得た。
【００４６】
変異を、ジデオキシ配列解析によって確認した（Ｓａｎｇｅｒ ｅｔ ａｌ．， Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． Ｕ．Ｓ．Ａ．， ｖｏｌ．７４， ｐｐ．５４６３−５４６７ （１９７７））。
図３で用いられたＤＮＡ配列および番号付けシステムを参照すると、＋１８８アミノ酸の位置をコードするコドンは、８１２〜８１４番の塩基対にある。ＰＣＲプライマーＡ＋およびＡ−は、７８４〜８０７番の塩基対に相当する。ＰＣＲプライマーＢ＋およびＢ−は、８１２〜８４４番の塩基対に相当する。ＰＣＲプライマーＬＡＡｆｓ５の５’末端は、５１８番の塩基対に相当する。ＰＣＲプライマーＰＣＲ Ｃｌａｌ−Ｓａｌｌの５’末端は、１３１７番の塩基対に相当する。Ａｓｐ７１８部位は、７２４番の塩基対に相当する。ＢｓｓＨＩＩ部位は、１０５３番の塩基対に相当する。
【００４７】
実施例３
Ｍ１５およびＮ１８８における変異をコードするプラスミドの構築
１５位のアミノ酸がメチオニンからスレオニンに置換されたｐＢＬａｐｒプラスミドを、米国特許出願第０８／１９４，６６４（ＰＣＴ公報ＷＯ９４／１８３１４）に従って構築した。このプラスミド（ｐＢＬａｐｒＭ１５Ｔ）を、ＳｆｉＩおよびＡｓｐ７１８で分解し、４７７ｂｐフラグメントをｐＨＰ．ＢＬにサブクローニングし、ｐＨＰ．Ｍ１５Ｔを製造した。上述した実施例１と同様の方法で、ｐＨＰ．Ｍ１５Ｔを、Ａｓｐ７１８およびＢｓｓＨＩＩで分解し、ゲルで精製し、該ゲルから溶離した。続いて、Ａｓｐ７１８−ＢｓｓＨＩＩを含む３３３ｂｐのフラグメント、および、ｐＨＰ．Ｎ１８８ＳからのフラグメントをｐＨＰ．Ｍ１５Ｔにサブクローニングし、プラスミドｐＨＰ．Ｍ１５Ｔ／Ｎ１８８Ｓを得た。同様の方法で、プラスミドｐＢＬａｐｒＭ１５ＬおよびｐＨＰ．Ｎ１８８Ｙを用いて、プライマーｐＨＰ．Ｍ１５Ｌ／Ｎ１８８Ｙを構築した。
【００４８】
実施例４
Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓのプラスミドによる形質転換、変異α−アミラーゼの発現および精製
実施例１〜３で説明したプラスミドによる形質転換の後、α−アミラーゼをＢａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓで発現させた。ｐＨＰ１３は、Ｅ．ｃｏｌｉやＢａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ中で複製させることができる。異なる変異を含むプラスミドを、Ａｎａｇｎｏｓｔｏｐｏｕｌｏｓ ｅｔ ａｌ．， Ｊ． Ｂａｃｔｅｒ．， ｖｏｌ．８１ ， ｐｐ．７４１−７４６ （１９６１）に説明されているように、Ｅ．ｃｏｌｉ株ＭＭ２９４を用いて構築し、該プラスミドを分離し、続いてＢａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓを形質転換した。Ｂａｃｉｌｌｕｓ株は二種のプロテアーゼ（Δａｐｒ、Δｎｐｒ）（例えば、Ｆｅｒｒａｒｉ等の米国特許第５，２６４，３６６号を参照）およびアミラーゼ（ΔａｍｙＥ）（例えば、Ｓｔａｈｌ ｅｔ ａｌ．， Ｊ． Ｂａｃｔｅｒ．， ｖｏｌ．１５８， ｐｐ．４１１−４１８ （１９８４）を参照）によって溶解させた。Ｍ１５Ｌ／Ｎ１８８Ｙを発現するＢａｃｉｌｌｕｓ株は、１％の不溶性スターチを含む寒天プレート上で、Ｍ１５Ｌを発現する株より大きい透明ゾーンを形成することから、デンプン分解活性が増強されていることがわかった。形質転換後、ｓａｃＵ（Ｈｙ）変異体（Ｈｅｎｎｅｒ ｅｔ ａｌ．， Ｂａｃｔｅｒ．， ｖｏ１．， １７０， ｐｐ．２９６−３００ （１９８８））を、ＰＢＳ−１で媒介される導入方法（Ｈｏｃｈ， Ｊ． Ｂａｃｔ．， ｖｏｌ．１５４， ｐｐ．１５１３−１５１５ （１９８３））によって導入した。
【００４９】
分泌されたアミラーゼを、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓの培養物から普通に以下のようにして回収した。培養物を、硫酸アンモニウムで２０％飽和に調節し、４℃で１時間攪拌した。遠心分離した後に得られた上清を、硫酸アンモニウム７０％飽和に調節し、４℃で１時間攪拌した。その上清を遠心分離した後に得られた沈殿を、ｐＨ６．０で５ｍＭの塩化カルシウムを含む５０ｍＭ酢酸ナトリウムに再溶解し、ろ過滅菌した。
【００５０】
実施例５
α−アミラーゼ活性を決定するための分析
可溶性基質分析（Ｓｏｌｕｂｌｅ Ｓｕｂｓｔｒａｔｅ Ａｓｓａｙ）：速度分析を、Ｍｅｇａｚｙｍｅ （Ａｕｓｔ．） Ｐｔｙ． Ｌｔｄ．が販売するエンド−ポイントアッセイキット（ｅｎｄ−ｐｏｉｎｔ ａｓｓａｙ ｋｉｔ）に基き行った。バイアル中の基質（ｐ−ニトロフェニルマルトヘプタオシド、ＢＰＮＰＧ７）を、１０ｍｌの滅菌水で溶解し、続いて分析緩衝液（５０ｍＭマレイン酸緩衝液、ｐＨ６．７、５ｍＭ塩化カルシウム、０．００２％Ｔｗｅｅｎ２０）で１：４に希釈した。分析は、２５℃のキュベット中で、１０μｌのアミラーゼを７９０μｌの基質に加えることによってなされた。７５秒間反応させた後に４１０ｎｍでの吸収が変化する速度を測定し、これを加水分解の速度とした。その結果によれば、０．２吸収単位／分（ａｂｓｏｒｐｔｉｏｎ ｕｎｉｔｓ／ｍｉｎ）の速度まで直線であった。
【００５１】
α−アミラーゼのタンパク質濃度は、Ｂｒａｄｆｏｒｄ， Ａｎａｌ． Ｂｉｏｃｈｅｍ．， ｖｏｌ． ７２， ｐ． ２４８ （１９７６）に基く、ウシ血清アルブミンを標準に用いた標準的なバイオラッドアッセイ（Ｂｉｏ−Ｒａｄ Ｌａｂｏｒａｔｏｒｉｅｓ）により測定された。
【００５２】
デンプン加水分解分析：デンプンに対するα−アミラーゼ活性は、デンプンがヨウ素を含む青色複合体を形成する能力、および、デンプンが加水分解されてデキストリン分子に分解されるときにその色が消失することによる分析方法によって決定された。α−アミラーゼ活性は、分解されて、デンプンがデキストリンの状態になったことを示す色の変化に必要な時間において決定された。
【００５３】
用いられた試薬は以下のとおりである。
リン酸緩衝液−リン酸２水素カリウム（３４０ｇ）および水酸化ナトリウム（２５．３ｇ）を、水に溶解し、〜２リットルに希釈した。該緩衝液を室温まで冷やし、ｐＨを６．２±０．１に調節した。該緩衝液をメスフラスコで２リットルに希釈した。
【００５４】
デンプン基質−１０ｇの可溶性リントナーデンプン（ｌｉｎｔｎｅｒ ｓｔａｒｃｈ）を、５０ｍｌの水に懸濁し、〜３００ｍｌの沸騰水中に投入した。その懸濁液を再び沸騰するまで加熱し、一定してかき混ぜながら５分間煮沸した。そのデンプン溶液を一定してかき混ぜながら室温まで冷やし、１２５ｍｌのリン酸緩衝液を添加した。溶液を５００ｍｌに水で希釈した。デンプン基質は、毎日新たに作成した。
【００５５】
保存ヨウ素溶液−ヨウ素の結晶（５．５ｇ）およびヨウ化カリウム（１１．０ｇ）を、水に溶解し、体積測定により２５０ｍｌに希釈した。その溶液を暗所に保存した。
【００５６】
希釈ヨウ素溶液−ヨウ化カリウム（２０ｇ）および２ｍｌの保存ヨウ素溶液を水に溶解し、体積測定により５００ｍｌに希釈した。溶液は、毎日新たに作成した。
【００５７】
酵素希釈溶液−塩化カルシウム（１１．１ｇ）を４リットルの水に溶解した。全ての試薬で用いられた水はいずれも蒸留水または脱イオン水であった。
α−アミラーゼサンプルを、１０〜１５ＬＵ／ｍｌ（以下で定義される）の範囲で、酵素希釈溶液で希釈した。多くの市販のα−アミラーゼ製品において、適切な希釈は、２０００倍であることがわかった。希釈ヨウ素溶液を５ｍｌずつ１３×１００ｍｍの試験管に分配し、１０ｍｌのデンプン基質を２３×２００ｍｍの試験管に入れた。全ての試験管を３０℃のウォーターバス中に置いた。特殊なα−アミラーゼカラーディスクを備えたＨｅｌｌｉｇｅ ｃｏｍｐａｒａｔｏｒ（カタログ番号 ６２０−ｓ５）を用いて、読み取りを行った。５ｍｌの希釈酵素（３０℃）を、デンプン基質と混合し、時間計測をはじめた。適切な時間間隔で、例えば、反応初期１分間および反応後期１５秒間の間隔で、酵素−基質混合物１ｍｌを希釈ヨウ素溶液を含む試験管に移送した。デンプンヨウ素溶液を混合し、１３ｍｍの精密角型試験管に移送し、その色を、Ｈｅｌｌｉｇｅ ｃｏｍｐａｒａｔｏｒの標準α−アミラーゼカラーディスクと比較した。エンドポイントの時間が近づいてきたときに、サンプルを０．２５分の間隔で分取した。
【００５８】
サンプルの色とカラーディスクの色とが合致するのに要する時間を記録し、活性（グラムまたはｍｌあたりの液化量（ｌｉｑｕｅｆｏｎ）において）を以下の式に従って計算した。
【００５９】
【数１】
ＬＵ／ｍｌまたはＬＵ／ｇ＝
式中、ＬＵは液化量単位（ｌｉｑｕｅｆｏｎ ｕｎｉｔ）であり、Ｖは酵素の量（５ｍｌまたはグラム）であり、ｔはデキストリン化する時間（分）であり、Ｄは希釈ファクター（希釈された酵素のｍｌまたはｇで希釈液量を割った値）である。
【００６０】
実施例６
熱安定性に関する、追加の変異α−アミラーゼの調製および試験
Ｂａｃｉｌｌｕｓ　ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓおよびＢａｃｉｌｌｕｓ　ａｍｙｌｏｌｉｑｕｅｌａｃｉｅｎｓは、Ｂａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓが生産するα−アミラーゼより安定性の劣るα−アミラーゼを生産する。これらのアミラーゼの配列から、Ｂａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓと比べてＢａｃｉｌｌｕｓ　ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓまたはＢａｃｉｌｌｕｓ　ａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓにおいて異なる残基を同定した。この分析から、Ｂａｃｉｌｌｕｓ　ｓｔｅａｌｏｔｈｅｒｍｏｐｈｉｌｕｓおよびＢａｃｉｌｌｕｓ　ａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ両方において対応する残基は同一であるがＢａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓとは異なるような５つの位置の１以上の位置で置換された、Ｂａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓの配列に基く変異α−アミラーゼを製造した。該５つの位置とは、Ｍ１５Ｔ／Ｈ１３３Ｙ／Ｎ１８８５／Ａ２０９Ｖと組み合わされたＡ３３Ｓ、Ａ５２Ｓ、Ｎ９６Ｑ、Ｓ１４８Ｎ、Ａ３７９Ｓであり、これらと、Ｍ１５Ｔ／Ｈ１３３Ｙ／Ｎ１８８５／Ａ２０９Ｖの置換のみを含む変異体と比較した。加えて変異体Ｓ８５Ｄを組み込み、これは、Ｂａｃｉｌｌｕｓ　ａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓのα−アミラーゼからのみ補充されたことを示す。望ましい変異体を得るために適切なＰＣＲプライマーを用いたことを除いては、実施例１〜４に記載の方法に従って変異体を製造した。
【００６１】
様々な変異体における熱失活の速度を、以下の方法によって測定した。アミラーゼ保存溶液を、２０ｍＭ酢酸アンモニウム、４ｍＭ　ＣａＣｌ_２、ｐＨ６．５の溶液中でよく透析した。安定性の測定のために、この保存溶液を、天然型アミラーゼの迅速な失活を誘導するように調製された溶液で５０倍超に希釈した。該溶液は、５０ｍＭ酢酸アンモニウム、５ｍＭ ＣａＣｌ_２、０．０２％Ｔｗｅｅｎ２０、ｐＨ４．９または４．８であり、最終濃度は３０〜５０μｇ／ｍｌであった。６個のエッペンドルフチューブに上記希釈された溶液を１００μｌずつ入れ、８２〜８３℃のウォーターバス中に置いた。一定時間でエッペンドルフチューブを取り出し、３０秒〜５分の間隔で測定し、失活を止めるために氷上においた。実施例５で説明した可溶性基質を用いて残存活性を分析した。活性の自然対数をインキュベート時間に対してプロットし、失活に関する速度定数を直線の傾きから得た。ｌｎ（２）を速度定数で割ることによって半減期を計算した。様々な変異体に関する結果を表１〜４に示す。
【００６２】
【表１】

【００６３】
【表２】

【００６４】
【表３】

【００６５】
【表４】

【００６６】
実施例７
より安定なセルラーゼの安定性の改良のための、より安定でないセルラーゼからの残基のより安定なセルラーゼへの補充
様々な種からのＴｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉ　ＥＧＩＩＩセルラーゼの相同体を得て、熱安定性を試験した。セルラーゼは、（Ａ）Ｈｙｐｏｃｒｅａ　ｓｃｈｗｅｉｎｉｔｚｉｉ、（Ｂ）Ａｓｐｅｒｇｉｌｌｕｓ　ａｃｕｌｅａｔｕｓ、（Ｃ）Ａｓｐｅｒｇｉｌｌｕｓ　ｋａｗａｃｈｉｉ、（Ｄ）Ｇｌｉｏｃｌａｄｉｕｍ　ｒｏｓｅｕｍ　（１）、（Ｅ）Ｇｌｉｏｃｌａｄｉｕｍ　ｒｏｓｅｕｍ（２）、（Ｆ）Ｇｌｉｏｃｌａｄｉｕｍ　ｒｏｓｅｕｍ　（３）および（Ｇ）Ｈｕｍｉｃｏｌａ　ｇｒｉｓｅａから得られた。上記のなかでもセルラーゼ（ａ）〜（ｆ）は、分析条件下でＥＧＩＩＩより熱的に安定ではない。セルラーゼ（ｇ）はＥＧＩＩＩより安定である。従って、これらのセルラーゼが並べられた配列（図１１に示す）から、より安定なセルラーゼに比べてより安定でないセルラーゼ中に存在する残基をピックアップすることができる。表の読み取りをわかりやすくするために、変異が記録された位置は太字で強調されている。続いてＥＧＩＩＩの変異体をこの情報に基き調製した。加えて、変異セルラーゼ（ｇ）は、ＥＧＩＩＩからの補充に基き調製された。
【００６７】
平衡円２色性を、Ａｖｉｖ市販の５ポジション熱電流セルホルダーを備えたＡｖｉｖ　６２ＤＳまたは６２ＡＤＳ分光光度計によって測定した。緩衝液は、酢酸でｐＨ８．０に調節した５０ｍＭビス−トリスプロパンおよび５０ｍＭ酢酸アンモニウムであった。各実験において最終的なタンパク質濃度は、５〜３０μＭの範囲であった。経路の長さ（ｐａｔｈ ｌｅｎｇｔｈ）０．１ｃｍのセル中でデータをとった。
【００６８】
２６５〜２１０ｎｍにおいてスペクトルを収集した。２１７ｎｍで、３０〜９０℃の範囲で熱変性させ、２℃毎にデータをとった。各温度における平衡時間は、０．１分であり、サンプルあたり４秒間データをとった。
【００６９】
ｐＨ８．０サンプルの残りを、５×４００μＬに分配した。２個のサンプルを酢酸でｐＨ５および７に調節し、他の２個を水酸化ナトリウムでｐＨ９および１０に調節した。全てのサンプルを上述したように同時に熱変性させた。
【００７０】
表５において、より安定でないセルラーゼからＥＧＩＩＩへの補充に基き調製された２４個の変異体、および、ＥＧＩＩＩからのＨｕｍｉｃｏｌａ ｇｒｉｓｅａから分離された相同体への補充に基き調製された３個の変異体を示す（表５におけるＨ．ｇｒｉｓｅａに対する番号付けは、図１１のＥＧＩＩＩの番号付けに基くことに留意すること）。これらのうち有意な数が、天然型分子（ＥＧＩＩＩまたはセルラーゼ（ｇ）のいずれか）より改良された安定性を有ししており、それらを表５において太字で示す。
【００７１】
【表５】

【図面の簡単な説明】
【図１】図１は、Ｂａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓのα−アミラーゼからのＡｓｎ１８８の特異的変異導入の間に有用な変異オリゴヌクレオチドを説明する。オリゴヌクレオチド構造を説明する図１〜１１において、太字の文字はオリゴヌクレオチドによって導入された塩基の変更を示し、下線はオリゴヌクレオチドによって導入された制限エンドヌクレアーゼ部位を示す。
【図２】図２は、変異オリゴヌクレオチドテンプレートのＰＣＲ処理に用いられるＰＣＲプライマーを説明する。
【図３】図３は、Ｇｒａｙ ｅｔ ａｌ．， Ｊ． Ｂａｃｔｅｒｉｏｌｏｇｙ， ｖｏｌ．１６６， ｐｐ．６３５−６４３ （１９８６）によって説明されている、Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ（ＮＣＩＢ ８０６１）のα−アミラーゼに関する遺伝子のＤＮＡ配列（配列番号３３）、および、翻訳産物から導き出されたアミノ酸配列（配列番号４１）を説明する。
【図４】図４は、Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓからの成熟α−アミラーゼ酵素のアミノ酸配列（配列番号３４）を説明する。
【図５】図５は、３種のバチルス属のα−アミラーゼの一次構造のアライメントを説明する。Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓのα−アミラーゼ（Ａｍ−Ｌｉｃｈ）（配列番号３５）は、Ｇｒａｙ ｅｔ ａｌ．， Ｊ． Ｂａｃｔｅｒｉｏｌｏｇｙ． ｖｏｌ．１ ６６， ｐｐ．６３５−６４３ （１９８６）によって説明されており、Ｂａｃｉｌｌｕｓ ａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓのα−アミラーゼ（Ａｍ−Ａｍｙｌｏ）（配列番号３６）は、Ｔａｋｋｉｎｅｎ ｅｔ ａｌ．， Ｊ． Ｂｉｏｌ Ｃｈｅｍ．， ｖｏｌ．２５８， ｐｐ．１００７−１０１３ （１９８３）によって説明されており、Ｂａｃｉｌｌｕｓ ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓのα−アミラーゼ（Ａｍ−Ｓｔｅａｒｏ）（配列番号３７）は、Ｉｈａｒａ ｅｔ ａｌ．， Ｊ． Ｂｉｏｃｈｅｍ．， ｖｏｌ．９８， ｐｐ．９５−１０３ （１９８５）によって説明されている。
【図６】図６は、プラスミドｐＨＰ１３を説明し、ここでＣｍ^Ｒはクロラムフェニコール耐性、Ｅｍ^Ｒはエリスロマイシン耐性、および、Ｒｅｐ　ｐＴＡ１０６０はプラスミドｐＴＡ１０６０からの複製のオリジンを意味する。
【図７】図７は、ｐＢＬａｐｒプラスミドを説明し、ここでＢＬ　ＡＡはＢａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓのα−アミラーゼ遺伝子、ａｐｒＥはａｐｒＥ遺伝子の領域をコードするプロモーターおよびシグナルペプチド、ＡｍｐＲはｐＢＲ３２２からのアンピシリン耐性遺伝子、ＣＡＴはｐＣｌ９４からのクロラムフェニコール耐性遺伝子を意味する。
【図８】図８は、Ｂａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓのα−アミラーゼをコードするｐＨＰ．ＢＬプラスミドを説明する。
【図９】図９は、Ｂａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓから得られるα−アミラーゼに対応する変異オリゴヌクレオチドを製造するのに用いられたＰＣＲ方法の模式図である。
【図１０】図１０は、Ｂａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓのα−アミラーゼにおけるシグナル配列−成熟タンパク質の結合（配列番号３８）、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ　ａｐｒＥのα−アミラーゼにおけるシグナル配列−成熟タンパク質の結合（配列番号３９）、および、ｐＢＬａｐｒ（配列番号４０）におけるＢａｃｉｌｌｕｓ　ｌｉｃｈｅｎｉｆｏｒｍｉｓのα−アミラーゼにおけるシグナル配列−成熟タンパク質の結合を説明する。
【図１１】図１１は、Ｔｒｉｃｈｏｅｒｍａ　ｒｅｅｓｅｉ（ＥＧＩＩＩ）（配列番号４１）、Ｈｙｐｏｃｒｅａ　ｓｃｈｗｅｉｎｉｔｚｉｉ（配列番号４２）、Ａｓｐｅｌｇｉｌｌｕｓ　ａｃｕｌｅａｔｕｓ（配列番号４３）、Ａｓｐｅｒｇｉｌｌｕｓ　ｋａｗａｃｈｉｉ（配列番号４４）、Ｈｕｍｉｃｏｌａ　ｇｒｉｓｅａ　（配列番号４５）、Ｇｌｉｏｃｌａｄｉｕｍ　ｒｏｓｅｕｍ（１）（配列番号４６）、Ｇｌｉｏｃｌａｄｉｕｍ　ｒｏｅｓｕｍ（２）（配列番号４７）、Ｇｌｉｏｃｌａｄｉｕｍ　ｒｏｓｅｕｍ（３）（配列番号４８）から得られた特定のセルラーゼに相当する、８個の相同なエンドヌクレアーゼ酵素の配列アライメントを説明する。番号付けは、ＥＧＩＩＩの番号付けに基づく。[0001]
Background of the Invention
The present invention relates to improved proteins, in particular enzymes having stability properties modified by the recruitment of residues from homologous or related proteins, and the development of such precisely improved proteins. For an effective way to enable.Background of the Invention
To increase the utility of proteins in various applications, methods for improving the functional properties of proteins are widely practiced. Such techniques include separating and selecting particular amino acid residues based on the properties of the particular amino acid and / or the structure of the protein, particularly those that are related or show a relationship based on experimental evidence. Often involves the use of protein engineering and site-directed mutagenesis. For example, U.S. Pat. No. 4,760,025 to Estell et al. Discloses an oxidation to improve stability by changing particularly oxidizable residues such as methionine, lysine, tryptophan and cysteine. Suggests modification of an unstable enzyme. U.S. Pat. No. 4,752,585 to Koths et al. Shows a method for improving the oxidative stability of a therapeutic protein by substituting conservative amino acids with methionine residues, each of which is susceptible to oxidation of chloramine T. U.S. Pat. No. 4,732,973 to Barr et al.₁-Substituting the active site methionine of antitrypsin with an oxidation stable amino acid.
[0002]
A further technique proposed so far is the homology modeling of proteins to near evolutionary proteins, which can provide a basis for protein engineering. See Siezen et al. , Protein Engineering, vol. 4, no. 7, pp. 719-737 (1991) discloses a method for improving the properties of proteases used in industrial processes by targeting or copying more stable enzyme features. Moreover, these techniques require that more desirable protein properties be imparted to less desirable proteins. However, in practice, the more desirable proteins are the targets for which it is desired to exhibit improved properties. Therefore, it is difficult to adjust such proteins to be more desirable. Therefore, researchers are looking at non-knowledge based techniques for improvement.
[0003]
Non-knowledge-based mutagenesis methods are required when information about the protein of interest is lacking or when knowledge-based techniques do not work. Techniques not based on typical knowledge include well-known methods such as random mutagenesis, error-prone PCR, and PCR template switching techniques, and furthermore, for example, US Patent No. 5,605,793 to Stemmer et al. Some have been developed recently, such as the shuffling described in the issue. Random mutagenesis has been used for many years, and the literature reports varying levels of success. However, random mutagenesis may succeed or fail due to the large number of potential variants. For example, a protein consisting of 300 amino acids becomes 300²⁰With a possible number of random variants of the order of It is not possible to prepare, express and screen such a large number of mutants, even using modern mass screening techniques. Constant evolution techniques, such as "gene shuffling" described in Stemmer or other molecular breeding techniques, are limited by the pool of available mutants. However, pools of mutants must be produced from either native or random mutations if the knowledge about the protein is inadequate. However, this pool of mutants may not provide the optimally focused pool of diverse species required for reasonable success.
[0004]
Considerable efforts have been made in the field of protein engineering to produce more stable enzymes. Nevertheless, with the vast amount of information on many important proteins, improved methods can be developed to intelligently focus pools of mutant molecules on specific effects in order to take full advantage of the range of biological diversity. Must be. As disclosed below, the present inventors have developed a mutagenesis technique based on new knowledge, and have excellent utilization in terms of producing a novel protein having improved performance, that is, functionality. Offer the possibility. By practicing the techniques of the present invention, researchers will be able to obtain a pool of mutants that facilitates the selection of desirable "successful" proteins.
[0005]
Summary of the Invention
Investigators who have faced the problem of improving the performance of a protein for a particular functional property often encounter serious problems with deciding to introduce appropriate mutations in the protein. Lack of knowledge of the crystal structures of many proteins and uncertainties involving low homology alignments with comparative proteins whose crystal structures are known are serious obstacles to knowledge-based mutagenesis methods. Furthermore, random mutagenesis techniques or other non-knowledge-based mutagenesis methods such as molecular breeding often screen large numbers of mutants without assurance that optimal mutagenesis is uniform. It is a way of knowing whether it will succeed or fail. However, researchers attempting to improve on certain characteristics often obtain families with an associated primary structure, many of which are less functional in function than the protein being sought. This occurs naturally due to the fact that the best protein is often chosen as the first target. However, while this "best protein" can produce significant commercial or industrial benefits from improvements, it has significant drawbacks. Thus, there remains a problem for researchers in determining how to modify a protein in a manner that improves other properties without diminishing the desired properties of the protein of interest. In this case, the scientists herein use an alignment of less desirable proteins, i.e., less stable or less active enzymes, to be accepted by the "best proteins" and to unjustify their good properties. It has been determined that it is possible to infer amino acid modifications that are highly successful in that they do not break. In fact, the chances of success are greatly enhanced, as reality has often already confirmed that the chosen modification will work in the intended process.
[0006]
Objects of the present invention include, for example, enhanced alkali stability, pH stability, thermal stability, enhanced oxidative stability, enhanced catalytic activity, and improved substrate or target binding, It is to provide a method for obtaining a protein with improved functional properties.
[0007]
It is a further object of the present invention to provide proteins, for example, with enhanced alkali stability, pH stability, thermal stability, enhanced oxidative stability, enhanced catalytic activity, and improved An enzyme that has substrate or target binding properties.
[0008]
The present invention comprises the steps of: (a) determining a primary amino acid sequence of a first protein and a second homologous protein, arranging the primary amino acid sequences, and producing a primary amino acid sequence alignment; (B) identifying different residues at corresponding positions between the first protein and the second homologous protein in the sequence alignment of the primary amino acid sequence; (C) preparing a mutant protein in the first protein in which substitution, deletion or addition is performed in correspondence with at least one of the different residues selected in step (b); Screening said mutant protein for improved performance for properties; (e) said mutant having improved performance. The step (e) of selecting and determining the identity of the protein, wherein said improved mutein differs in at least one residue from both said first protein and said second homologous protein, Wherein the first protein has an improved function with respect to the desired property; and a method for improving the desired property of the first protein. Preferably, an alignment of the first protein with two or more second homologous proteins is made and the substitution selected in step (c) is performed at the particular position selected for mutation by the second homologous protein. Are selected from residues that are present in significant or whole parts of Preferably, the improved protein has improved stability properties with respect to alkali, pH, heat or oxidation compared to the protein corresponding to the precursor amino acid sequence. Also preferably, said protein is an enzyme, more preferably α-amylase, lipase, cellulase or protease.
[0009]
In other embodiments of the invention, (a) aligning the primary amino acid sequences of at least two comparison proteins, one of the comparison proteins having less desirable performance for the desired property; (b) Identifying positions where the residues differ between at least two comparative enzymes and determining the residues present at the positions identified in the comparative protein with less desirable performance for the desired property; (c) in step (b) Selecting one or more determined residues; (d) replacing the selected residue in the first protein at a position corresponding to the position in the comparison protein where the residue was selected in step (c). Modifying the first protein by substitution, deletion or addition, provided that the target protein is the first protein and the comparative protein. Differing in at least one residue from the first, wherein the improved first protein has an improved function for the desired property, wherein the method for producing a protein of interest has improved properties as compared to the first protein. Provided.
[0010]
In a preferred embodiment, the comparison is performed with two or more second homologous proteins, and residues that are present in all or only a few of the less stable second homologous proteins are supplemented to the first protein to improve Produce protein.
[0011]
In an embodiment of the composition of the invention, an improved protein comprising a modification according to the method of the invention is obtained.
An advantage of the present invention is that a protein having more desirable properties can be produced by a simple technique of juxtaposing and analyzing the sequences of two homologous proteins having different properties.
[0012]
Another advantage of the present invention is that the pool of potential mutants can be significantly reduced to the number of mutants that are more easily screened as opposed to methods that are not based on mutant knowledge.
[0013]
Yet another advantage of the present invention is that no information on the tertiary structure of the protein to be improved is required, and only a sequence alignment of homologous proteins is required.
Yet another advantage of the present invention is that superior proteins can be further improved based on information obtained from less desirable proteins, and thus can be utilized to develop such improved proteins. The accumulation of information can be increased.
[0014]
Detailed description
By "expression vector" is meant a DNA construct, including a DNA sequence operably linked to appropriate control sequences capable of expressing the DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to regulate such transcription, a sequence encoding an appropriate mRNA ribosome binding site, and a sequence that controls transcription and translation termination. , May be included. If expression is desired in a Bacillus host, a preferred promoter is the Bacillus subtilis aprE promoter. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and operate independently of the host genome, or in some cases, integrate into the genome itself. Although plasmids and vectors may be used interchangeably, as plasmids are the most commonly used form of vector at present, the present invention has equivalent functions and is well known or well known in the art. It is intended to include other expression vector forms such as
[0015]
By "host strain" or "host cell" is meant a suitable host for an expression vector comprising DNA encoding a protein according to the present invention. A host cell useful in the present invention is generally a prokaryotic or eukaryotic host and includes any transformable microorganism capable of achieving expression of a given protein according to the present invention. Those of skill in the art will be familiar with appropriate expression and secretion mechanisms, including suitable host cells, for use with a particular protein. For example, a host strain of the same species or genus from which the particular protein is derived is suitable, for example, for an α-amylase from the genus Bacillus, a suitable host cell may be a Bacillus strain. In this case, α-amylase-negative Bacillus strains (deletion of the gene) and / or Bacillus strains lacking α-amylase and protease (ΔamyE, Δapr, Δnpr) are preferably used. A host cell is transformed or transfected with a vector constructed using recombinant DNA technology. Such a transformed host cell can replicate a protein-encoding vector or a modified product thereof (mutant), or can express a desired protein.
[0016]
"Recombinant protein" is defined as a mutant DNA sequence encoding a sequence in which one or more amino acids have been substituted, inserted or deleted, as compared to a naturally produced protein or protein precursor sequence. , A protein produced by modifying a DNA sequence encoding a naturally produced protein or a protein precursor. As used herein, the term precursor protein (or enzyme) means the parent protein to be modified, rather than the precursor just before the specific expression in the chemical reaction. Thus, although it is a precursor that defines a modification, the actual alteration or modification will have its basis in alterations in the DNA encoding the precursor, which will subsequently transform and express the DNA. Secretes the protein product that contains the modification.
[0017]
The improved protein according to the present invention comprises an amino acid sequence derived from the amino acid sequence of a precursor protein (or also referred to herein as a "first protein"), wherein certain modifications in the precursor protein are provided herein. It is something like The precursor protein can be a naturally occurring protein or a recombinant protein. The amino acid sequence of the improved protein is derived from the amino acid sequence of the precursor protein by substitution, deletion or insertion of one or more amino acids in the precursor amino acid sequence. Such modifications are generally of the precursor DNA sequence encoding the amino acid sequence of the precursor protein, rather than manipulating the precursor protein itself. Suitable methods for such manipulation of the precursor DNA sequence include the methods disclosed herein and U.S. Patent Nos. 4,760,025 and 5,185,258 of the same owner. , Incorporated herein by reference.
[0018]
By "desired properties" is meant any properties for which it is desired to improve the protein. Such desirable properties may be, for example, functional properties such as heat, pH, alkali, oxidation or chemical stability, catalytic activity, substrate binding, receptor binding, allergenicity, antimicrobial activity, or any other desired protein. Including features.
[0019]
"Primary amino acid sequence alignment" means an alignment of the primary sequences of at least two proteins to obtain a significant degree of sequence identity. Various alignment algorithms are appropriate to those of skill in the art and exist in the form of commercially available software that is used routinely. These programs provide inputs for various algorithm parameters that may act to obtain a result. However, the present invention does not require a choice for any particular sequence alignment algorithm, and the choice can generally optimize parameters. Generally, the alignment is considered an approximation, that is, one should choose from it by advantageously providing a slightly different pool of variants using various methods to optimize the alignment. Moreover, because the advantage of the present invention is to produce a target pool of mutant proteins to select for improved proteins, the present invention is not limited to programs or parameters used to determine alignments. Should be equally applicable.
[0020]
According to one embodiment of the invention, the protein is modified as follows to improve its performance for the desired property. The primary amino acid sequence of the modified protein, termed the "first protein", is aligned with the primary amino acid sequence of a comparative "second homologous protein" that has less desirable performance for the desired property. From the alignment, the positions at which the residues differ are determined, and the residues present at those positions in the second homologous protein sequence. Subsequently, at least one residue selected in the previous step is incorporated into the amino acid sequence of the first protein. As the inventor has now confirmed with surprise, modified proteins produced in this manner have a significant probability of having improved performance for desirable properties. Thus, the pool of muteins produced in this manner has various combinations of the identified residues substituted, deleted or added, and possibly comprises an improved pool of proteins, followed by a "success" Can be selected from among them.
[0021]
In variations, an alignment of more than two primary amino acid sequences can be obtained. In this embodiment, the first protein is aligned with the primary amino acid sequence of two or more second homologous proteins, but the primary amino acid sequence of each second homologous protein may be different from that of the first protein. Corresponding to the protein. From this alignment, residues present in two or more second homologous proteins at positions where there is a difference between the primary amino acid sequence of the first protein and the primary amino acid sequences of two or more second homologous proteins select. In another method of expanding the pool of available mutants, each of the variations between the first and second homologous proteins can be used as a basis for obtaining a mutant. Other preferred selectable methods limit the number of mutants available to an optimal number to successfully confer improved functional properties to the selected mutant protein, such that each of the second homologous proteins has the same identity. Only those differences that contain residues are selected.
[0022]
In one variation of the above method, the alignment is provided by two or more second homologous proteins that are not directly compared to the first protein. For example, determining which residues are different by aligning two comparative homologous proteins, each of which can be aligned with the first protein. The resulting alignment is then analyzed to determine the residues present in one or more comparison proteins with less desirable performance, and then to replace those residues at corresponding positions in the first protein. , Delete or add. In this embodiment, the comparative protein need not be superior or inferior to the first protein, and the residues selected to modify the protein of interest have less desirable performance for the desired properties. Or a homologous comparison protein. In addition, the primary amino acid sequences of more than two second homologous proteins can be aligned and residues selected from them based on the performance of the various second homologous proteins with respect to the desired properties.
[0023]
Surprisingly and surprisingly, Applicants have prepared an alignment of two or more homologous or related proteins, the residues present at various positions in the protein having less desirable performance for the desired properties. It has been found that by matching, a protein with improved functional properties may be obtained. For example, comparing a first protein with a second homologous protein having less desirable properties for a desired functional property, identifying different residues, preparing a pool of variants based on the different mutations described above, and By selecting a variant that is improved for the desired property relative to the first protein, an improvement that is advantageous in the desired property may be obtained with a significant probability of such modification. That is, one of the outstanding uses of the present invention is to produce relatively small and easy-to-handle mutant pools (compared to a pool of random mutants), which are significantly improved compared to the first protein. Proteins with different functional properties can be selected therefrom. Furthermore, such a result can be obtained without detailed structure and function information.
[0024]
The aligned proteins do not necessarily have to have comparable activity or function. However, in a preferred embodiment, the proteins have comparable activities, ie, similar biological activities, functions, catalytic activities, or other criteria commonly used to classify specific proteins. However, the present invention is based on the determination of the positions identified by reality, where residues can be present in the structure of a protein without significantly affecting its value on the host organism and are compared. Not all features of a given protein need be the same, only some homology. "Substantially homologous" means that the proteins are conserved, i.e., have significant amino acid identity, so that their sequences are meaningfully arranged and can define major structural, functional or catalytic sites. It is to have at a certain level. Preferably, the first and second homologous proteins contain at least 30%, preferably 50% sequence identity, more preferably 70% sequence identity, most preferably 85% sequence identity.
[0025]
In a preferred embodiment, the protein is an enzyme. The enzymes include any of the five major classes of enzymes: hydrolases, oxidoreductases, transferases, lyases or ligases. Specific examples of enzymes that can benefit from the present invention include amylase, lipase, cellulase, protease, hemicellulase, glucoamylase, esterase, lactase, polygalacturonase, β-galactosidase, ligninase, oxidase, peroxidase, glucose. Includes isomerase or any closely related enzyme or low stability homolog.
[0026]
Α-amylase is described below as a typical example of the method and constitution of the present invention. As used herein, “α-amylase” means, for example, one having an enzymatic activity that cleaves or hydrolyzes α (1-4) glycoside bonds in starch, amylopectin or amylose polymer. α-amylases include naturally produced α-amylases and recombinant α-amylases. Preferred α-amylases in the present invention are those derived from Bacillus species, in particular Bacillus licheniformis, Bacillus amyloliquefaciens, or Bacillus stearothermophilus, as well as, for example, Aspergillus (ie, A. e. E., Such as Aspergillus (i.e., A. e. E. Ger.) And A. e. From α-amylases. Therefore, the α-amylase in the present invention is derived from the precursor α-amylase. The precursor α-amylase is produced from any source capable of producing α-amylase. Preferred sources of α-amylase are prokaryotes or eukaryotes, including fungi, bacteria, plants or animals. The precursor α-amylase is preferably produced by the genus Bacillus, more preferably produced by Bacillus licheniformis, Bacillus amyloliqueefaciens, or Bacillus stearothermophilus, most preferably produced by Bacillus licheniformis.
[0027]
So far, homology has been found between almost all endoamylases across sequenced plants, mammals and bacteria (Nakajima et al., Appl. Microbiol. Biotechnol., Vol. 23, pp. 355). 360 (1986); Rogers, Biochem. Biophys. Res. Commun., Vol. 128, pp. 470-476 (1985); Janessek, Eur. J. Biochem., Vol. 224, pp. 519-524. ). In a certain type of Bacillus amylase, there are four regions exhibiting particularly high homology, and as shown in FIG. 5, the underlined portion is a region exhibiting high homology. Sequence alignments have also been used to map relationships between Bacillus endoamylases (Feng et al., J. Molec. Evol., Vol. 35, pp. 351-360 (1987)). The relative sequence homology between Bacillus stearothermophilus and Bacillus licheniformis is about 66%, and between Bacillus licheniformis and Bacillus amyloliquefaciens is about 81%, which is about 80%. , Protein Engineering, vol. 3, No. 3, pp. 181-191 (1990). To establish homology to the primary structure, the amino acid sequence of the precursor α-amylase is compared directly with the primary sequence of Bacillus licheniformis α-amylase, especially that the sequence is invariant in all known α-amylases. Direct comparison with known groups of residues (see, eg, FIG. 7). In addition, porcine pancreatic α-amylase (Buisson et al, EMBO Journal vol. 6, pp. 3909-3916 (1987); Qian et al., Biochemistry, vol. 33, pp. 6284-6294 (1994); Larson. al., J. Mol. Biol., vol. 235, pp. 1560-1584 (1994)): Taka-amylase A of Aspelgillus oryzae (Matsuura et al., J. Biochem. Vol. 95, pp. 697-702 (1984)); It is also possible to determine equivalent residues by tertiary structure analysis of the crystal structure reported for acid α-amylase from Niger (Boel et al., Biochemistry, vol. 29, pp. 6244-6249 (1990)). Yes, the previous two structures are similar, and barley α-amylase (Vallee et al., J. Mol. Biol., Vol. 236, pp. 368-371 (1994); Kadziola, J. Mol. Biol., Vol.239, pp.104-121 (1994)), and it is also possible to determine equivalent residues by tertiary structure analysis of the crystal structure reported. Several preliminary studies have been published (Suzuki et al, J. Biochem., Vol. 108, pp. 379-381 (1990): Lee et al. Arch. Biochem. Biophys, vol. 291, pp. 255-257 (1991); Chang et al, J. Mol. Biol., Vol.229, pp.235-238 (1993): Mizuno et al., J. Mol.Biol., Vol.234, pp. 1282-1283 (1993)), there is only one published structure for the α-amylase of Bacillus licheniformis (Machius et al., J. Mol. Biol. Vol. 246, pp. 545-549 (1995). )). However, many researchers have reported that the common super-secondary structure between glucanase (MacGregor et al., Biochem. J., vol. 259, pp. 145-152 (1989)) and α-amylase and Ultra-secondary structure common to enzymes that metabolize other starches (Jaspersen, J. Prot. Chem. Vol. 12, pp. 791-805 (1993); MacGregor, Starke, vol. 45, pp. 232). -237 (1993)) and sequence similarity between α-amylase and an enzyme having a similar supersecondary structure (Janecek, FEBS Letters, vol. 316, pp. 23-26 (1993); et al, J. Prot. Chem., vol. 12, predicts the pp.509-514 (1993)). The structure for the enzyme of Bacillus stearothermophilus has been modeled for the structure of Taka-amylase A (Holm et al., Protein Engineering, vol. 3, pp. 181-191 (1990)). The four highly conserved regions shown in FIG. 7 are according to the Bacillus licheniform numbering system, including His + 105, Arg + 229, Asp + 231, His + 235, Glu + 261, and Asp + 328, and many residues considered to be part of the active site. (Matsuura et al., J. Biochem. (Tokyo), vol. 95, pp. 697-702 (1984): Buisson et al., EMBO Journal, vol. 6, pp. 3909-3916 (1987). Vihinen et al., J. Biochem., Vol. 107, pp. 267-272 (1990)). The crystal structure of the bacterial amylase hybrid enzyme has been published in PCT publication WO 96/23874.
[0028]
As mentioned above, the α-amylases from Bacillus licheniformis, Bacillus stearothermophilus, Bacillus amyloliquefaciens and Bacillus subtilis all have significant homology. However, under conditions of industrial starch liquefaction, such as, for example, high temperature (above 90 ° C.), low pH (pH 4-6), and low calcium, α-amylase obtained from Bacillus licheniformis has the most satisfactory performance. Show. Nevertheless, even Bacillus licheniformis α-amylase is susceptible to undesirable instability under liquefaction conditions producing more stable and desirable alternatives. Therefore, much work has been done on Bacillus licheniformis producing molecules to obtain the desired enzymatic properties for use in liquefaction processes. By aligning the sequence of the α-amylase from Bacillus licheniformis with the sequence of the α-amylase from Bacillus stearothermophilus or Bacillus amyloliquefaciens, it is possible to identify residues that differ between homologs. Subsequently, according to the present invention, the position at which the residue is different in the α-amylase from Bacillus stearothermophilus or Bacillus amyloliquefaciens as compared to the α-amylase from Bacillus licheniformis is changed from the α-amylase from the Bacillus licheniformis to the α-amylase from Bacillus licheniformis. Choose for Preferably, the substituted residues are in fact identical to the residues present in the α-amylase of either Bacillus stearothermophilus or Bacillus amyloliquefaciens. More preferably, the substituted residues are the same as those present in the α-amylase of both Bacillus stearothermophilus and Bacillus amyloliquefaciens.
[0029]
The residues for replacing Bacillus licheniformis specifically identified here are different from the residues at the corresponding positions in the α-amylase of Bacillus licheniformis and / or Bacillus stearothermophilus, especially A33, A52, S85, N96, H133, and S148. Particularly preferred substitutions for these residues are selected from the residues present in the α-amylase from Bacillus amyloliquefaciens and Bacillus stearothermophilus, and are A33S, A52S, N96Q, H133Y, S148N, A209V, A369S, and A379S, and A379S. And all are thought to contribute to stability. In addition, the A85D mutant is recruited only from Bacillus amyloliquefaciens, which also provides stability benefits. The above residues may be altered in combination with other modifications that provide performance benefits.
[0030]
The proteins improved according to the invention exhibit improved performance characteristics, thereby making them particularly useful in various applications where proteins are commonly used and improved stability is desired. For example, enzymes, including α-amylase, exhibit improved thermostability, improved pH stability, and / or improved oxidative stability according to the present invention. The enhanced thermal stability will be useful in extending the shelf life of the products containing them and in high temperature applications. Enhanced oxidative stability or enhanced performance is particularly desirable in cleaning products, and storage of enzymes in the presence of bleach, perborate, percarbonate or peracid used in such cleaning products. Extend life. The α-amylases of the present invention are particularly useful in starch-related processes, especially starch liquefaction where oxidation and thermostability are particularly important. Cellulases having improved stability are also disclosed in the present invention and are particularly useful, for example, in biomass reduction, cleaning products and textile treatment compositions.
[0031]
A further embodiment of the present invention is a DNA encoding a protein according to the present invention, and an expression vector comprising such a DNA. The DNA sequences may be expressed by treating them with expression control sequences in a suitable expression vector using conventional techniques and transducing the expression vector into a suitable host. Various types of host / expression vector combinations may be used to express the DNA sequences of the present invention. Useful expression vectors include, for example, chromosomal or non-chromosomal segments useful for this purpose, and synthetic DNA sequences such as various known plasmids and phages. In addition, a variety of expression control sequences can generally be used in these vectors. For example, with α-amylase obtained from Bacillus, Applicants have found that a preferred expression control sequence for Bacillus transformants is the aprE signal peptide derived from Bacillus subtilis.
[0032]
A variety of host cells are also useful for expressing the DNA sequences of the present invention. These hosts are, for example, It may include well-known eukaryotic and prokaryotic hosts, such as E. coli strains, Pseudomonas, Bacillus Streptomyces, various fungi, yeast and animal cells. A host that expresses the protein of the present invention extracellularly is preferred because purification and downstream processing are easy. Expression and purification of the improved proteins of the present invention can be made by art-recognized means for performing such a process.
[0033]
The following examples are set forth but should not be construed as limiting the scope of the claims. Abbreviations used herein, especially the three letter or one letter designations for amino acids, are described in Dale, J. et al. W. , Molecular Genetics of Bacteria, John Wiley & Sons, (1989) Appendix B.
[0034]
Example
Example 1
Plasmid pHP. Construction of BL
The α-amylase gene shown in FIG. 3 was cloned from Bacillus licheniformis NCIB8061 (Gray et al., J. Bacteriology, vol. 166, pp. 635-643 (1986)). The 1.72 kb PstI-SstI fragment encodes at least three residues of the signal sequence, the entire mature protein, and the terminal region, and was subcloned into M13mp18. A synthetic oligonucleotide cassette designed to contain the transcriptional regulatory ends of Bacillus amyloliquefaciens subtilisin (Wells et al., Nucleic Acid Research, vol. 11, pp. 7911-7925 (1983)) in the form shown below. A synthetic terminator was inserted between the BclI and SstI sites.
[0035]
Embedded image

[0036]
The pBLapr plasmid was constructed to contain the gene for Bacillus licheniformis α-amylase. As illustrated in FIG. 7, pBLapr binds the ampicillin resistance gene from pBR322 and the chloramphenicol resistance gene from pC194, the aprE promoter and the gene encoding the α-amylase of Bacillus licheniformis (“BL AA”). 6.1 kb plasmid. The aprE promoter was composed of a 660 bp HindIII-PstI fragment encoding the promoter and signal sequence of the alkaline protease of Bacillus subtilis. The Pstl site was removed and a SfiI site was added near the aprE / BL @ AA junction. The BLΔAA gene is contained in the 1720 bp PstI-SstI fragment described above. In the experiments described herein, pBLapr is composed of an SfiI site adjacent to the 5 'end of the start site of the coding sequence for mature amylase. In particular, the EcoRI-SstII fragment was subcloned from pBLapr to M13BM20 (Boehringer Mannheim) at the 5 'end of the pBLapr structure to obtain a coding strand template for the mutant oligonucleotide shown below.
[0037]
Embedded image

[0038]
This primer was introduced at the SfiI site (underlined). Thereby, the correct form could be screened by the presence of this particular restriction site. The EcoRI-SstH fragment was subcloned back into the pBLapr vector to obtain a version of the plasmid with an SfiI site.
[0039]
Plasmid pHP13 (Haima et al., Mol. Gen. Genet., VOL. 209, pp. 335-342 (1987)) (FIG. 6) was digested with restriction enzymes EcoRI and HindIII, and the resulting vector was poly Purified on acrylamide gel and eluted. Plasmid pEhapr was digested with HindIII and Asp718, and in a separate incubation, digested with Asp718 and EcoRI and gel purified. The two bands, HindIII-Asp718 (1203 bp) and Asp718-EcoRI (1253 bp), were purified on a gel, eluted from the gel, ligated to the vector by 3-way ligation and ligated into the vector pHP. BL was obtained. The plasmid was used for α-amylase expression (FIG. 8).
[0040]
Example 2
Construction of a plasmid encoding an α-amylase containing a substitution for asparagine 188
Mutant primers encoding substitution of Asn188 ("N188") by each naturally occurring amino acid were synthesized and are shown in FIG. 1 (SEQ ID NOs: 4-22). Α-Amylase gene variants encoding these mutations were produced by PCR using the PCR primers shown in FIG. 2 (SEQ ID NOS: 23-32) by the method summarized in FIG.
[0041]
Step (1): The extended (61 bp) double-stranded DNA was obtained using the mutant primer as a template for the PCR primers PCR @ A + and PCR @ B-. Each contained a different amino acid substitution at position 188, and all but N188M contained different restriction sites. First, the PCR primers were annealed at 35 ° C. for 5 minutes, followed by DNA extension with Taq polymerase at 75 ° C. for 1 minute. The double-stranded DNA was then dissociated at 95 ° C. for 1 minute, followed by an annealing step and an extension step. The cycle of dissociation, annealing and extension lasted a total of 30 cycles.
[0042]
Step (2): Upstream and downstream DNA at position 188 were synthesized in separate PCR reactions. As template, pBLapr was used, and as PCR primers, LAAfs5 (SEQ ID NO: 27) and PCR @ A- (SEQ ID NO: 24) were used for upstream DNA, and PCR @ B + (SEQ ID NO: 25) and PCR @ Cla for downstream DNA. -SalI (SEQ ID NO: 28) was used. The DNA was dissociated at 95 ° C for 1 minute, annealed at 45 ° C for 3 minutes, and extended at 68 ° C for 3 minutes. The upstream part is 290 bp and the downstream part is 498 bp. This method was repeated for 18 cycles using pfu polymerase. The same PCR method was used in steps (3) and (4).
[0043]
Step (3): The upstream portion of the DNA described in Step (2) was added to the double-stranded mutant primer described in Step (1). Primers LAAfs5 (SEQ ID NO: 27) and PCRΔB- (SEQ ID NO: 26) were used. Primers are designed so that there is a 24 bp overlap between the three templates, to which two pieces of DNA can be attached.
[0044]
Step (4): The downstream portion of the DNA shown in step (2) was combined with the product of step (3) to obtain the desired product. The 24 bp overlap between the two PCR products allows binding. The primers used were LAAfs5 (SEQ ID NO: 27) and PCRΔClaI-SalI (SEQ ID NO: 28).
[0045]
Step (5): Specific restriction sites, namely Asp718 and BssHII, are upstream and downstream of 188 sites, respectively. The PCR product of interest was digested with Asp718 and BssHII, and the 333 bp fragment was separated by polyacrylamide gel electrophoresis. Subcloned into the BL vector, pHP. N188X was obtained.
[0046]
Mutations were confirmed by dideoxy sequence analysis (Sanger et al., Proc. Natl. Acad. Sci. USA, vol. 74, pp. 5463-5467 (1977)).
Referring to the DNA sequence and numbering system used in FIG. 3, the codon encoding the +188 amino acid position is at bases 812-814. PCR primers A + and A- correspond to base pairs 784-807. PCR primers B + and B- correspond to base pairs 812-844. The 5 'end of the PCR primer LAAfs5 corresponds to the 518th base pair. The 5 'end of the PCR primer PCR Clal-Sall corresponds to the 1317 base pair. The Asp718 site corresponds to the 724 base pair. The BssHII site corresponds to base position 1053.
[0047]
Example 3
Construction of plasmids encoding mutations at M15 and N188
A pBLapr plasmid in which the amino acid at position 15 was replaced with methionine to threonine was constructed according to US patent application Ser. No. 08 / 194,664 (PCT Publication WO 94/18314). This plasmid (pBLaprM15T) was digested with SfiI and Asp718, and the 477 bp fragment was digested with pHP. BL. M15T was produced. In the same manner as in Example 1 described above, pHP. M15T was digested with Asp718 and BssHII, purified on a gel and eluted from the gel. Subsequently, a 333 bp fragment containing Asp718-BssHII and pHP. The fragment from N188S was converted to pHP. M15T and subcloned into plasmid pHP. M15T / N188S was obtained. In a similar manner, plasmid pBLaprM15L and pHP. N188Y and the primer pHP. M15L / N188Y was constructed.
[0048]
Example 4
Transformation with Bacillus subtilis plasmid, expression and purification of mutant α-amylase
After transformation with the plasmids described in Examples 1-3, α-amylase was expressed in Bacillus subtilis. pHP13 is E. coli. coli and Bacillus subtilis. Plasmids containing the different mutations are described in Anagnostopoulos et al. , J. et al. Bacter. , Vol. 81 pp. 741-746 (1961). E. coli strain MM294, the plasmid was isolated and subsequently transformed into Bacillus subtilis. Bacillus strains comprise two proteases (Δapr, Δnpr) (see, eg, US Pat. No. 5,264,366 to Ferrari et al.) And an amylase (ΔamyE) (eg, Stahl et al., J. Bacter., Vol. 158, pp. 411-418 (1984)). The Bacillus strain expressing M15L / N188Y formed a clear zone larger than the strain expressing M15L on agar plates containing 1% insoluble starch, indicating that the amylolytic activity was enhanced. After transformation, a sacU (Hy) mutant (Henner et al., Bacter., Vol. 1, 170, pp. 296-300 (1988)) was introduced into a PBS-1 mediated transfection method (Hoch, J. Bact. , Vol.154, pp.1513-1515 (1983)).
[0049]
Secreted amylase was recovered from a culture of Bacillus subtilis as usual. The culture was adjusted to 20% saturation with ammonium sulfate and stirred at 4 ° C. for 1 hour. The supernatant obtained after centrifugation was adjusted to 70% ammonium sulfate saturation and stirred at 4 ° C. for 1 hour. The precipitate obtained after centrifuging the supernatant was redissolved in 50 mM sodium acetate containing 5 mM calcium chloride at pH 6.0, and sterilized by filtration.
[0050]
Example 5
Assays for determining α-amylase activity
Soluble Substrate Assay: Kinetic analysis was performed using Megazyme (Aust.) Pty. Ltd. The analysis was performed based on an end-point assay kit sold by the company. The substrate (p-nitrophenylmaltoheptaoside, BPNPG7) in the vial was dissolved in 10 ml of sterile water, followed by analysis buffer (50 mM maleate buffer, pH 6.7, 5 mM calcium chloride, 0.002% Tween 20). The analysis was performed by adding 10 μl amylase to 790 μl substrate in a 25 ° C. cuvette. After reacting for 75 seconds, the rate at which the absorption at 410 nm changes was measured and this was taken as the rate of hydrolysis. The results showed that the curve was linear up to a rate of 0.2 absorption units / min.
[0051]
The protein concentration of α-amylase can be determined according to Bradford, Anal. Biochem. , Vol. 72, p. 248 (1976) using a standard Bio-Rad assay using bovine serum albumin as a standard (Bio-Rad Laboratories).
[0052]
Starch hydrolysis analysis: Α-amylase activity on starch is determined by the ability of starch to form a blue complex containing iodine, and by an analytical method by the loss of its color when starch is hydrolyzed and broken down into dextrin molecules. Was. The α-amylase activity was determined at the time required for a color change indicating that the starch had been degraded and the starch was in a dextrin state.
[0053]
The reagents used are as follows.
Phosphate buffer-potassium dihydrogen phosphate (340 g) and sodium hydroxide (25.3 g) were dissolved in water and diluted to ２2 liter. The buffer was cooled to room temperature and the pH was adjusted to 6.2 ± 0.1. The buffer was diluted to 2 liters in a volumetric flask.
[0054]
Starch substrate-10 g of soluble lintner starch was suspended in 50 ml of water and poured into ~ 300 ml of boiling water. The suspension was heated again to boiling and boiled for 5 minutes with constant stirring. The starch solution was cooled to room temperature with constant stirring and 125 ml of phosphate buffer was added. The solution was diluted to 500 ml with water. The starch substrate was made fresh daily.
[0055]
Stock iodine solution-iodine crystals (5.5 g) and potassium iodide (11.0 g) were dissolved in water and diluted to 250 ml by volume measurement. The solution was stored in the dark.
[0056]
Diluted iodine solution-potassium iodide (20 g) and 2 ml of the stock iodine solution were dissolved in water and diluted to 500 ml by volume measurement. Solutions were made fresh daily.
[0057]
Enzyme diluent-calcium chloride (11.1 g) was dissolved in 4 liters of water. The water used for all reagents was either distilled or deionized water.
The α-amylase sample was diluted with the enzyme diluent solution in the range of 10-15 LU / ml (defined below). For many commercial α-amylase products, the appropriate dilution was found to be 2000-fold. 5 ml of the diluted iodine solution was dispensed into 13 × 100 mm test tubes, and 10 ml of the starch substrate was placed in a 23 × 200 mm test tube. All tubes were placed in a 30 ° C. water bath. Readings were performed using a Heli comparator (catalog number 620-s5) equipped with a special α-amylase color disc. 5 ml of the diluted enzyme (30 ° C.) was mixed with the starch substrate and timed. At appropriate time intervals, for example at intervals of 1 minute early in the reaction and 15 seconds late in the reaction, 1 ml of the enzyme-substrate mixture was transferred to a test tube containing the diluted iodine solution. The starch iodine solution was mixed and transferred to a 13 mm precision square test tube and its color was compared to a standard alpha-amylase color disc from Hellig comparator. Samples were taken at 0.25 minute intervals as the endpoint time approached.
[0058]
The time required for the color of the sample to match the color of the color disc was recorded and the activity (in liquefons per gram or ml) was calculated according to the following formula:
[0059]
(Equation 1)
LU / ml or LU / g =
Where LU is the liquefaction unit, V is the amount of enzyme (5 ml or gram), t is the dextrinization time (minutes), and D is the dilution factor (diluted enzyme). (Diluent volume divided by ml or g).
[0060]
Example 6
Preparation and testing of additional mutant α-amylase for thermostability
Bacillus stearothermophilus and Bacillus amyloliquelaciens produce α-amylases that are less stable than those produced by Bacillus licheniformis. From the sequences of these amylases, different residues were identified in Bacillus stearothermophilus or Bacillus ｍ amyloliquefaciens compared to Bacillus licheniformis. From this analysis, it can be seen that in both Bacillus stearothermophilus and Bacillus amyloliquefaciens, the corresponding residue in the Bacillus licheniformis α-mutase α-mutated in the amino acid sequence of the Bacillus licheniformis α-mutase was substituted at one or more of the 5 positions such that the corresponding residues were identical but different from Bacillus licheniformis. Was manufactured. The five positions are A33S, A52S, N96Q, S148N, A379S in combination with M15T / H133Y / N1885 / A209V, which were compared to a mutant containing only the M15T / H133Y / N1885 / A209V substitution. . In addition, mutant S85D was incorporated, indicating that it was only recruited from Bacillus amyloliquefaciens α-amylase. Mutants were prepared according to the methods described in Examples 1-4, except that the appropriate PCR primers were used to obtain the desired mutant.
[0061]
The rate of heat inactivation in the various mutants was measured by the following method. The amylase stock solution was prepared using 20 mM ammonium acetate, 4 mM CaCl₂And dialyzed well in a solution of pH 6.5. For stability measurements, this stock solution was diluted more than 50-fold with a solution prepared to induce rapid inactivation of native amylase. The solution was 50 mM ammonium acetate, 5 mM CaCl₂, 0.02% Tween 20, pH 4.9 or 4.8, and the final concentration was 30-50 μg / ml. 100 μl of the diluted solution was placed in each of six Eppendorf tubes and placed in a water bath at 82 to 83 ° C. The Eppendorf tubes were removed at regular intervals, measured at intervals of 30 seconds to 5 minutes, and placed on ice to stop inactivation. Residual activity was analyzed using the soluble substrate described in Example 5. The natural log of activity was plotted against incubation time, and the rate constant for inactivation was obtained from the linear slope. Half-life was calculated by dividing In (2) by the rate constant. The results for various variants are shown in Tables 1-4.
[0062]
[Table 1]

[0063]
[Table 2]

[0064]
[Table 3]

[0065]
[Table 4]

[0066]
Example 7
Replenishment of more stable cellulases with residues from less stable cellulases for improved stability of more stable cellulases
Homologs of Trichoderma reesei EGIII cellulase from various species were obtained and tested for thermostability. Cellulases are (A) Hypocrea schweinitzii, (B) Aspergillus aculeatus, (C) Aspergillus kawachiii, (D) Gliocladium roseum (1), (E) Glioc radium (F) ) Obtained from Humicola grisea. Among the above, cellulases (a)-(f) are less thermally stable than EGIII under analytical conditions. Cellulase (g) is more stable than EGIII. Therefore, from the sequence in which these cellulases are arranged (shown in FIG. 11), it is possible to pick up residues that are present in a less stable cellulase than in a more stable cellulase. The positions where the mutations were recorded are highlighted in bold to make the table easier to read. Subsequently, variants of EGIII were prepared based on this information. In addition, mutant cellulases (g) were prepared based on supplementation from EGIII.
[0067]
Equilibrium circular dichroism was measured by an Aviv # 62DS or 62ADS spectrophotometer equipped with an Aviv commercial 5-position thermoelectric cell holder. The buffers were 50 mM bis-trispropane and 50 mM ammonium acetate adjusted to pH 8.0 with acetic acid. The final protein concentration in each experiment ranged from 5 to 30 μM. Data was taken in a cell with a path length of 0.1 cm.
[0068]
Spectra were collected at 265-210 nm. Thermal denaturation at 217 nm in the range of 30-90 ° C and data were taken every 2 ° C. The equilibration time at each temperature was 0.1 minute and data was collected for 4 seconds per sample.
[0069]
The rest of the pH 8.0 sample was dispensed into 5 × 400 μL. Two samples were adjusted to pH 5 and 7 with acetic acid and the other two were adjusted to pH 9 and 10 with sodium hydroxide. All samples were heat denatured simultaneously as described above.
[0070]
In Table 5, 24 mutants prepared based on recruitment of less stable cellulase to EGIII, and three mutants prepared based on recruitment of homologs isolated from Humicola grisea from EGIII (Note that the numbering for H. grisea in Table 5 is based on the EGIII numbering in Figure 11). Significant numbers of these have improved stability over native molecules (either EGIII or cellulase (g)) and are shown in Table 5 in bold.
[0071]
[Table 5]

[Brief description of the drawings]
FIG. 1 illustrates mutant oligonucleotides useful during the specific mutagenesis of Asn188 from Bacillus の licheniformis α-amylase. In FIGS. 1-11, which illustrate the oligonucleotide structure, bold letters indicate base changes introduced by the oligonucleotide, and underlines indicate restriction endonuclease sites introduced by the oligonucleotide.
FIG. 2 illustrates PCR primers used for PCR treatment of a mutant oligonucleotide template.
FIG. 3 shows the results of Gray et al. , J. et al. Bacteriology, vol. 166 pp. 635-643 (1986) describes the DNA sequence of the gene for α-amylase of Bacillus licheniformis (NCIB 8061) (SEQ ID NO: 33) and the amino acid sequence derived from the translation product (SEQ ID NO: 41). I do.
FIG. 4 illustrates the amino acid sequence of the mature α-amylase enzyme from Bacillus licheniformis (SEQ ID NO: 34).
FIG. 5 illustrates the alignment of the primary structure of three Bacillus α-amylases. Bacillus licheniformis α-amylase (Am-Lich) (SEQ ID NO: 35) was obtained from Gray et al. , J. et al. Bacteriology. vol. 166, pp. 635-643 (1986), and the α-amylase of Bacillus amyloliquefaciens (Am-Amylo) (SEQ ID NO: 36) is described in Takkinen et al. , J. et al. Biol Chem. , Vol. 258, pp. 1007-1013 (1983), and the α-amylase of Bacillus stearothermophilus (Am-Stearo) (SEQ ID NO: 37) is described in Ihara et al. , J. et al. Biochem. , Vol. 98 pp. 95-103 (1985).
FIG. 6 illustrates plasmid pHP13 where Cm^RIs chloramphenicol resistant, Em^RIs erythromycin resistance, andRep pTA1060 is the origin of replication from plasmid pTA1060.Means
FIG. 7 illustrates the pBLapr plasmid, where BLΔAA is the Bacillus licheniformis α-amylase gene, aprE is the promoter and signal peptide encoding the region of the aprE gene, AmpR is the ampicillin resistance gene from pBR322, CAT means chloramphenicol resistance gene from pCl94.
FIG. 8 shows the pHP.RTM. Encoding α-amylase of Bacillus licheniformis. The BL plasmid will be described.
FIG. 9 is a schematic diagram of a PCR method used to produce a mutant oligonucleotide corresponding to α-amylase obtained from Bacillus licheniformis.
FIG. 10 shows the signal sequence-mature protein binding in Bacillus licheniformis α-amylase (SEQ ID NO: 38), the signal sequence-mature protein binding in Bacillus subtilis aprE α-amylase (SEQ ID NO: 39), and , PBLapr (SEQ ID NO: 40) illustrates the signal sequence-mature protein binding in Bacillus licheniformis α-amylase.
FIG. 11 shows that Trichoerma reesei (EGIII) (SEQ ID NO: 41), Hypocrea schweinititzi (SEQ ID NO: 42), Aspergillus aculeatus (SEQ ID NO: 43), Aspergillus kawachii (SEQ ID NO: 44), Humse (SEQ ID NO: 44) 8 homologous ends corresponding to a specific cellulase obtained from Gliocladium @ roseum (1) (SEQ ID NO: 46), Gliocladium @ roesum (2) (SEQ ID NO: 47), Gliocladium @ roseum (3) (SEQ ID NO: 48) The sequence alignment of the nuclease enzyme will be described. Numbering is based on EGIII numbering.

Claims (14)

Process:
(A) determining the primary amino acid sequence of the first protein and the second homologous protein, aligning the primary amino acid sequences, and producing a primary amino acid sequence alignment, wherein the second homologous protein is more desirable than the first protein for desirable properties; Have no properties;
(B) in the sequence alignment of the primary amino acid sequence, identifying different residues at corresponding positions between the first protein and the second homologous protein;
(C) preparing a mutant protein in the first protein, wherein the mutant protein has been substituted, deleted or added corresponding to at least one of the different residues selected in step (b);
(D) screening the mutant protein for improved performance with respect to functional properties;
(E) selecting and determining the identity of said mutein having improved performance, wherein the improved mutein comprises at least one residue from both the first and second homologous proteins. Wherein the improved first protein has an improved function for a desired property;
A method for improving desirable properties of a first protein, comprising: 2. The method of claim 1, wherein the primary amino acid sequence of the first protein is aligned with two or more second homologous proteins. Step (c) comprising preparing a mutein comprising a substitution, deletion or addition selected from only those residues in which at least 50% of the corresponding positions of the second homologous protein are identical in the alignment. Item 3. The method according to Item 2. 2. The method according to claim 1, wherein the protein is an enzyme. 2. The method of claim 1, wherein the second homologous protein has at least 30% sequence identity with the first protein. 6. The method of claim 5, wherein the second homologous protein has at least 85% sequence identity with the first protein. 2. The method of claim 1, wherein the improved protein has improved oxidation, alkali, heat or pH stability, improved substrate binding, or improved catalytic activity. 2. The method of claim 1, wherein the alignment in step (a) is obtained by comparing the primary amino acid sequence of the first protein with the primary amino acid sequences of at least two different second homologous proteins. 9. The method of claim 8, wherein the variant is prepared in step (b) based on residues present at positions in the primary amino acid sequence of at least two second homologous proteins. 10. The method of claim 9, wherein the variants are based on residues that are equally present in the primary amino acid sequence of each second homologous protein. (A) aligning the primary amino acid sequences of at least two comparison proteins, one of the comparison proteins having less desirable performance for the desired property;
(B) identifying positions where the residues differ between the at least two comparative enzymes and determining the residues present at the positions identified in the comparative protein having less desirable performance for the desired property;
(C) selecting one or more residues determined in step (b);
(D) modifying the first protein by substituting, deleting or adding the selected residue in the first protein at a position corresponding to the position in the comparison protein where the residue was selected in step (c) To do;
Wherein the protein of interest differs in at least one residue from both the first protein and the comparative protein, wherein the improved first protein has an improved function with respect to the desired property. A method for producing a target protein having improved desirable properties as compared to one protein. 12. The method of claim 11, wherein two or more proteins are produced by steps (c) and (d), and wherein the protein of interest is selected from the two or more proteins. A protein prepared by the method of claim 1. A protein prepared by the method of claim 11.

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